Modulation Of Fruit sOftening By Antisense Suppression

Mol Breeding (2011) 27:375–383 DOI 10.1007/s11032-010-9438-9

Modulation of fruit softening by antisense suppression of endo-b-1,4-glucanase in strawberry
Young Koung Lee • In-Jung Kim

Received: 19 August 2009 / Accepted: 2 April 2010 / Published online: 16 April 2010 Ó Springer Science+Business Media B.V. 2010

Abstract To test the effect of endo-b-1,4-glucanase (EGase) in strawberry, weproduced transgenic strawberry plants that contained sense and antisense cDNA encoding strawberry EGase under the CaMV 35S promoter (CaMV35S-P) and the strawberry fruit dominant ascorbate peroxidase (APX) promoter. Independent transgenic lines were generated and the firmness of the fruit was characterized after harvest. Interestingly, transgenic lines that harbored the cDNA antisense under theCaMV 35S promoter were not generated, but transgenic lines that contained sense EGase cDNA (FraCel1) under the CaMV 35S promoter and sense and antisense EGase cDNA under the APX promoter were successfully obtained using a tissue culture system. Reverse transcriptase
Electronic supplementary material The online version of this article (doi:10.1007/s11032-010-9438-9) contains supplementary material,which is available to authorized users.
Y. K. Lee Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea I.-J. Kim (&) Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju 690-756, Korea e-mail: ijkim@jejunu.ac.kr I.-J. Kim Research Institute of Subtropical Agriculture and Biotechnology, Jeju NationalUniversity, Jeju 690-756, Korea

polymerase chain reaction (RT-PCR) analysis revealed that the steady-state transcript levels of CaMV35S-P:FraCel1 sense transgenic lines were dramatically increased in fruit evaluated at all stages, as well as in the leaves. Moreover, real-time PCR analysis demonstrated that the steady-state transcript level of FraCel1 in independent transgenic lines that containedantisense FraCel1 under the APX promoter was reduced in fruit during the turning and red stages when compared with wild-type strawberries. These results were consistent with the firmness of strawberries that contained the antisense FraCel1 under the APX promoter, which was 19–36% greater in the turning stage and 22–25% greater in the red stage when compared to wild-type strawberries. Taken together,these findings indicate that fruit-specific down-regulation of the EGase gene using the APX promoter is an effective technique for increasing fruit firmness in strawberries. Keywords Ascorbate peroxidase Á Endo-b-1,4-glucanase Á Firmness Á Fragaria 9 ananassa Á Transgenic strawberry

Introduction Storage and transportation following fruit harvest has long been studied in fruiting vegetablesbecause the overall quality of fruit is determined by how long it can be maintained and preserved. Indeed, preservation

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is an important aspect of economics in agronomy. Molecular breeding technology that produces fruit goods that better withstand storage and transportation was first commercially developed in the Flavr Savr tomato. In climacteric fruit,the polygalacturonase (PG) gene product involved in fruit softening has been examined in transgenic plants. The results of these experiments have shown that PG mRNA accumulation in transgenic plants was suppressed by constitutive expression of the antisense PG gene, and that this suppression had an effect on fruit softening during ripening (Sheehy et al. 1988; Smith et al. 1990; Pear et al. 1993).This approach was easily applied to climacteric fruits that are primarily regulated by the production of ethylene during fruit maturation. However, this method has not been directly adapted to nonclimacteric fruits that are not directly controlled by ethylene, such as strawberries, citrus fruit and grapes (Davies et al. 1997; Llop-Tous et al. 1999). In strawberry, the preservation of cell wall...